Immune inductor and effector sites in the upper airways of cattle and influence of site of antigen expression on induction of mucosal immunity. THIS G

Abstract

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Technical Summary

Respiratory mucosal surfaces are the main portal of entry for many pathogens of cattle, a number of which are a significant threat to animal welfare and result in severe economic loss. Examples include Mycobacterium bovis, the causative agent of bovine tuberculosis, and foot and mouth disease virus (FMDV). Pathogens that invade via the respiratory mucosa represent an enormous challenge for the development of vaccines that either prevent the pathogen from attaching to and colonising respiratory mucosa or from penetrating and replicating within respiratory epithelial cells. There is increasing evidence from studies in mice that intranasal vaccination is superior to vaccination at other sites in eliciting protection within the respiratory tract. Intranasal vaccination is, therefore, an attractive strategy for the control of infectious diseases in cattle. However, safe and effective intranasal vaccines have proved difficult to develop. Progress in the development of strategies for induction of protective immunity in the upper airways of cattle are hampered by a lack of detailed knowledge of mucosal immune inductor and effector sites, the role of different antigen-presenting cells in the nasopharynx, the type of vaccine construct most appropriate for stimulating mucosal immunity and the availability of bio- containment facilities needed for the study of some bovine pathogens (M. bovis and FMDV). This project aims to exploit recent advances in the manipulation of the genome of negative-stranded viruses, culture techniques for well- differentiated, bovine airway epithelial cells and the characterisation of bovine dendritic cells (DCs) to: (i) Identify the anatomic location and functional potential of bovine nasopharyngeal immune inductor and effector sites (ii) Identify antigen-presenting cells in bovine upper airway tissues (iii) Determine if targeting foreign antigens to different sites within polarised airway epithelial cells influences the induction of mucosal immunity The project will utilise recombinant bovine respiratory syncytial virus (rBRSV) expressing green fluorescent protein or the E2 protein of bovine virus diarrhoea virus (BVDV) as tools to determine the ability of bovine airway epithelial cells to act as antigen-presenting cells, in vitro; to investigate the effects of virus infection of airway epithelial cells on maturation of DCs; and to characterise DC subsets migrating from tissue explants of bovine nasal-associated lymphoid tissue, tonsils, and respiratory epithelium, following virus infection in vitro. The relevance of the findings obtained from these in vitro studies to the induction of mucosal immunity, in vivo, will be determined by investigating mucosal and systemic immune responses induced following intranasal vaccination of calves with rBRSVs expressing BVDV E2 protein at different sites within polarised airway epithelial cells. The information generated from this project will allow a more targeted approach to the development of intranasal vaccines that will be of broad relevance for a variety of bovine pathogens. In addition, these studies may lead to the development of laboratory methods for evaluating mucosal vaccines, thereby reducing the number of animals used in research.

Bovine respiratory syncytial virus (BSRV) is a major cause of respiratory disease in calves and is closely related to human RSV. Bovine viral diarrhoea virus (BVDV) is responsible for disease that affects the respiratory, reproductive, immune and enteric systems of cattle. These diseases are a huge problem globally for the livestock industry and a mucosally targeted, bivalent vaccine which prevents infection and spread of these viruses would be valuable. We investigated the influence of the site of expression of the BVDV E2 protein on induction of immune responses using recombinant (r)BRSV expressing wild-type and mutant forms of the E2 protein targeted to different sites within polarised epithelial cells. Calves vaccinated intranasally with rBRSV expressing the E2 protein at the basolateral surface induced higher levels of E2-specific serum antibodies than animals vaccinated with rBRSV expressing E2 at the apical membrane, intracellularly or when secreted from cells. Targeting E2 to the basolateral surface also induced E2-specific IgA antibodies in respiratory secretions without compromising RSV-specific responses. Calves vaccinated intranasally with rBRSV expressing E2 targeted to the basolateral surface were completely protected against challenge with BRSV, and were also protected against nasopharyngeal excretion of BVDV. These studies highlight the importance of the site of expression of foreign proteins in polarised epithelial cells on priming of immune responses.Dendritic cells are central in the initiation of immune responses and also contribute to the induction and maintenance of peripheral tolerance. The induction of tolerance to inert environmental antigens is essential at mucosal surfaces such as the respiratory tract. Dendritic cells have been identified underlying mucosal epithelia in rodent models and in the respiratory tract as immature or semi mature cells with differing cytokine profiles. There is little or no information regarding bovine respiratory tract DC. We showed that bovine tracheal DC can be induced to migrate from tracheal epithelial explants during culture with GMCSF and are identified as MHC class IIhigh cells which express moderate levels of CD208 together with CD40, CD80 and CD86. Differential expression of CD26 and signal regulatory protein a (SIRPa or CD172a) has been observed suggesting that subsets of DC exist with potentially divergent functions. A comparison of surface marker expression on migrated tracheal DC was compared to DC from lymph nodes and tonsils in the bovine upper respiratory tract. There were significantly more SIRPa positive DC in the trachea and lung which increased during BRSV infection. Analysis of tissues from BRSV infected calves showed SIRPa positive DC were more likely to contain BRSV antigen compared to SIRPa negative cells. Both populations were able to present antigen to drive T cell proliferation and IFNy production predominantly from CD8+ T cells.

Exploitation Route

Identification and characterisation of DC within the bovine respiratory tract will allow greater understanding of their functions in both the steady state and following infectious challenge or mucosal vaccination.

Sectors

Agriculture, Food and Drink

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